Improved fusion technique. I. Human umbilical cord serum, a new and potent growth promoter, compared with other B cell and hybridoma activators

Westerwoudt, Regine J.; Blom, Joke; Naipal, A.; Rood, Jon J. van

doi:10.1016/0022-1759(83)90110-2

Cited by 20 publications

(4 citation statements)

References 12 publications

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“…Three days after the last injection, the spleen cells from an immunized animal were used in hybridoma production according to the method of Kennett [18]. After fusion, the cells were gently resuspended in HIT growth medium (DME, 20% fetal calf serum, insulin, 100 ~tM hypoxanthine, 16 txM thymidine, 1 mM sodium pyruvate, 4% human cord serum, 0.22% sodium bicarbonate supplemented with 1 x nonessential amino acids), dispensed into 96 well plates, and incubated at 37 °C for 24 h [6,40]. HIT with 1 pm aminopterin (HIAT) was added to each well after 24 h. Fourteen days after the fusion the HIAT medium was removed and HIT medium added.…”

Section: Hybridoma Productionmentioning

confidence: 99%

Topographical analysis of the G virion of Aleutian mink disease parvovirus with monoclonal antibodies

Barnard

Johnson

1992

Archives of Virology

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The topography of the Aleutian mink disease parvovirus (ADV) G virion was analyzed with monoclonal antibodies and polyclonal antiserum. There was homology between the two major structural proteins as others have previously reported. Trypsin treatment of the virion with subsequent immunoblotting revealed that VP2 represents the main peptide on the exterior of virion and that VP1 is probably embedded within the capsid. Additional analyses of the trypsin-treated virions showed that VP2 is responsible for binding complement and that it also represents the structural part of the virion that binds to cellular receptors. A third protein, p34, was detected that might represent a third structural polypeptide because of its many unique epitopes relative to the other peptides detected.

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Section: Hybridoma Productionmentioning

confidence: 99%

Topographical analysis of the G virion of Aleutian mink disease parvovirus with monoclonal antibodies

Barnard

Johnson

1992

Archives of Virology

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“…The cellswere gentlyresuspended in Iscove'smedium (Gibeo) supplemented with 10% fetal calf serum (v/v, Gibco), penicillin (100 Vlml), streptomycin (100 ug/m), Gibco), glutamine 2xlO-3 M, Gibco), Na-pyruvate (10-3 M, Gibco), 2-mercaptoethanol (5x10-~M, Sigma), hypoxanthine (10--' M, Sigma), thymidine (1.6x 10-~M, Sigma)and aminopterin (11100, Gibco). For fusion with SR lymphocytes,a portion ofthe cellsuspensionwasseeded in96-well plates (Nunc) withmouseperitoneal macrophages [18] asfeeder, part with5% human umbilical cord serum [19] and part withoutanyfeeder. Allfurther fusionswereperformed byusing5% human umbilical cord serum as feeder.…”

Section: Fusion Procedures and Limitingdilutionmentioning

confidence: 99%

Production of Stable Human‐Mouse Hybridomas Secreting Monoclonal Antibodies against Rh D and c Antigens

et al. 1993

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Peripheral blood lymphocytes from donors immunized against Rh antigens were fused with mouse myelomas and heteromyelomas in order to obtain human-mouse hybridomas secreting antibodies specific for these antigens. Three cell lines secreting anti-D IgG and two secreting anti-c IgM were stabilized and produced immunoglobulins for several months. These human monoclonal antibodies were evaluated as reagents for Rh phenotyping. Their complementary activity towards weak D and partial D antigens is examined.

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“…After 1 week of culture, the HAT medium was replaced by hypoxanthine-thymidine (HT) medium containing 10% FCS. After cell proliferation, the supernatant of the cell cultures was screened for antibodies and the antibody producing hybridomas were cloned by limiting dilutions in HT medium supplemented with 10% FCS and 5% human umbilical cord serum [31]. Monoclonal antibodies were tested with 125I-labelled insulin and C-peptide in order to define the molecule moiety containing the epitope.…”

Section: Preparation Of Monoclonal Antibodies Against Human Proinsulinmentioning

confidence: 99%

Influence of affinity of antibodies upon their detection by liquid phase radiobinding assay and solid phase enzyme linked immunosorbent assay. Demonstration using monoclonal antibodies raised against rDNA human proinsulin

et al. 1991

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Hybridomas producing proinsulin antibodies were cloned by limiting dilution of cell cultures obtained by fusion of splenocytes of immunized mice with immortal myeloma cells. Some proinsulin monoclonal antibodies crossreacted with labelled insulin but none did with labelled C-peptide indicating that the involved epitopes were at one of the insulin/C-peptide junctions or included in the insulin moiety. Hybridoma supernatants were assayed for IgG concentration by a solid phase assay and for ligand binding by a radiobinding assay and an enzyme linked immunosorbent assay. The half-life of immune complexes formed with radioligand was measured and, as expected, correlated with affinity as measured by the method of Scatchard. Antibody titres determined by enzyme linked immunosorbent assay did not correlate to those measured by radiobinding assay. IgG concentration correlated to enzyme linked immunosorbent assay titres but not to radiobinding assay titres. Finally, a significant correlation was found between radiobinding assay titre and the product of enzyme linked immunosorbent assay titre by the period of immune complexes. It is concluded that, except for very low affinity antibodies, enzyme linked immunosorbent assay is a capacity assay whereas radiobinding assay is influenced by both antibody concentration and affinity. The former assay is thus best suited to detecting low affinity antibodies whereas the latter is more efficient in the presence of low levels of high affinity antibodies.

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Improved fusion technique. I. Human umbilical cord serum, a new and potent growth promoter, compared with other B cell and hybridoma activators

Cited by 20 publications

References 12 publications

Topographical analysis of the G virion of Aleutian mink disease parvovirus with monoclonal antibodies

Topographical analysis of the G virion of Aleutian mink disease parvovirus with monoclonal antibodies

Production of Stable Human‐Mouse Hybridomas Secreting Monoclonal Antibodies against Rh D and c Antigens

Influence of affinity of antibodies upon their detection by liquid phase radiobinding assay and solid phase enzyme linked immunosorbent assay. Demonstration using monoclonal antibodies raised against rDNA human proinsulin

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